The induction of neuronal differentiation in PC12 cells by nerve growth factor (NGF) is a model system for studying how an external stimulus causes a cell to terminally differentiate. In order to understand the mechanism by which NGF triggers neuronal differentiation, we are studying the regulation of the peripherin gene, a neural specific intermediate filament protein which is transcriptionally induced with a late time course when NGF stimulates PC12 cells to differentiate into neurons. Previously we have defined proximal and distal positive elements in the peripherin promoter which are necessary for induction by NGF, as well as a negative regulatory element (NRE) which has a functional role in repressing peripherin activity in undifferentiated and non-neuronal cells. DNA mobility shift assays demonstrate that the protein complex binding to the NRE is altered during NGF-induced neuronal differentiation. We have proposed a two-step model of induction of peripherin-by NGF in which dissociation of a repressor from the protein complex at the NRE, coupled with a positive signal from the distal positive element, results in derepression of the gene. This proposal will focus on the negative regulation of peripherin by repressor proteins binding to the NRE. A major goal will be to understand how NGF-mediated mechanisms relieve repression of peripherin in differentiated PC12 cells. The hypothesis that a repressor protein is released from the NRE during NGR-mediated differentiation will be directly tested by the DNA affinity precipitation assay. The repressor protein will be purified and/or cloned in order to determine how NGF modifies its expression and activity. In addition, possession of the cloned repressor protein will facilitate studies using in vitro transcription to define the level of action of the repressor protein, as well as structural studies identifying the repressor domain of the protein. These experiments will further our understanding of mechanisms of negative transcriptional control. Finally, the effect of mutation at the NRE on the expression pattern of a peripherin promoter-lacZ fusion gene in transgenic mice will indicate the in vivo importance of the NRE for the neuronal-specific control of peripherin expression. Understanding the mechanism by which NGF induces neural-specific gene expression in PC12 cells may suggest approaches to trigger terminal differentiation in related tumor cells such as neuroblastoma.